Difference between revisions of "Part:BBa K316006:Experience"

m
m
 
Line 18: Line 18:
 
[[Image:HMStime2.PNG|thumb|right|250px|Graph shows production of HMS over time after catechol addition at time 0 minutes. Different colored curves represent different substrate concentrations added at time 0min in cell cultures.]]
 
[[Image:HMStime2.PNG|thumb|right|250px|Graph shows production of HMS over time after catechol addition at time 0 minutes. Different colored curves represent different substrate concentrations added at time 0min in cell cultures.]]
  
−
==GFP-XylE expression with or without TEV protease==
+
==GFP-XylE expression in the presence or absence of TEV protease==
 
[[Image:All 3.jpg|thumb|center|400px]]
 
[[Image:All 3.jpg|thumb|center|400px]]
 
The graph presents the data acquired in an experiment to compare HMS production of cell cultures that:
 
The graph presents the data acquired in an experiment to compare HMS production of cell cultures that:
Line 30: Line 30:
  
  
−
The results show that TEV protease cleaves the GFP-XylE fusion protein, producing active XylE protein. This can be seen by comparing the blue (XylE) and the green (GFP-XylE in presence of TEV) curve data, as they show similar rates of production of HMS colored product. The difference in the absorbance plateau value is due to slightly different initial concentrations of catechol substrate.  
+
The results show that the GFP-XylE fusion is far less able to tetramerise to form active XylE enzymes and break down catechol. TEV protease (expressed in the cell with the GFP-XylE) cleaves the GFP-XylE, producing active XylE protein. This can be seen by comparing the blue (XylE) and the green (GFP-XylE in presence of TEV) curve data, as they show similar rates of production of HMS colored product.  
 +
 
 +
The plateauing effect is caused by depletion of the catechol substrate and differences in the height of the absorbance plateaus value is due to slightly different initial concentrations of catechol substrate (both approximately 0.2mM).  
 +
  
 
|};
 
|};
  
 
<!-- DON'T DELETE --><partinfo>BBa_K316006 EndReviews</partinfo>
 
<!-- DON'T DELETE --><partinfo>BBa_K316006 EndReviews</partinfo>

Latest revision as of 20:57, 5 November 2010

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K316006

  • This part can be used as an inducible reporter enzyme.GFP was added to the N-terminus of C2,3O monomer. The C2,3O enzyme is an obligate homotetramer where all 4 subunits are required for its activity. By adding GFP at the N-terminus of the each monomer, tetramerization of the enzyme is blocked, thus rendering it inactive. This modification is important as it makes the system inducible. The cleavage of the GFP by TEV protease is the input inducer signal, means that Catechol dioxygenase monomers are released so that they can form the active tetramer.

User Reviews

UNIQ12a9462fc305aecb-partinfo-00000000-QINU

•••••

Imperial College iGEM 2010

  • There is about a 10-fold reduction in the rate at which the protein product of GFP-XylE gene construct catalyses the reaction in comparison to the wild type XylE gene.
Graph shows production of HMS (yellow product) over time after catechol addition at time 0 minutes. Different curves represent different catechol concentration added to the cell cultures
Graph shows production of HMS over time after catechol addition at time 0 minutes. Different colored curves represent different substrate concentrations added at time 0min in cell cultures.

GFP-XylE expression in the presence or absence of TEV protease

All 3.jpg

The graph presents the data acquired in an experiment to compare HMS production of cell cultures that:


1)Express XylE gene (blue)

2)Express GFP-XylE in the absence of TEV (red)

3)Express GFP-XylE along with TEV protease (green)


The results show that the GFP-XylE fusion is far less able to tetramerise to form active XylE enzymes and break down catechol. TEV protease (expressed in the cell with the GFP-XylE) cleaves the GFP-XylE, producing active XylE protein. This can be seen by comparing the blue (XylE) and the green (GFP-XylE in presence of TEV) curve data, as they show similar rates of production of HMS colored product.

The plateauing effect is caused by depletion of the catechol substrate and differences in the height of the absorbance plateaus value is due to slightly different initial concentrations of catechol substrate (both approximately 0.2mM).


;

UNIQ12a9462fc305aecb-partinfo-00000002-QINU